Study On Cloning And Function Analysis Of Terpenoid Biosynthetic Genes In Actinomycetes

Poultry processing industries and slaughterhouses produced several million tons of feathers waste annually.These keratinaceous wastes are important resources containing valuable proteins and amino acids and the main components are keratin.The high stability of keratin depend on high degree of cross-linkages by disulfide bonds.Microbial degradation is becoming an attractive approach to manage keratinous wastes.In this paper,the soil samples were collected from local poultry farm inYang Ling,China.13 bacterial isolates were selected on basal medium containing feather powder as the sole carbon and nitrogen source.According to 16 S rDNA PCR-RFLP for the 13 isolated bacteria,their ability of feather degradation were tested.One strain ZJT01 with high keratinolytic activity were gotten which could completely degrade 10g/L feathers in 7 day.16 S rDNA sequence similarity analysis showed that strain ZJT01 belongs to the genus Arthrobacter with the highest sequence similarity to Arthrobacter nitroguajacolicus G2-1T(100%).Combined with morphological and physiological characteristics,it was then designated as Arthrobacter sp.ZJT01.The characteristies for keratinase of strain ZJT01 were investigated.The results show: the optimum conditions for fermentarion were temperatures of 30? and ferment time of 72 hours.The optimum p H and temperature for keratinase activity were pH 8 and 35?,respectively.Isopropanol,DMSO,CaCl2 and KCl were inhibit the enzyme activity,MgCl2 were increased enzyme activity.But the enzyme was inhibited by PMSF,so it indicates that the enzyme were the serine protease.We have used genome shuffling to enhanced keratinase activity.The mutant strains were screened by diethyl sulfate(DES)and nitrosoguanidine(NTG)mutagenesis and then genome shuffling allows the recombination of multi-parental crossing through a recursive protoplast fusion,it can improve feather-degrading efficiency.In this study,ZJT01 bacteria was established the suitable conditions of DES and NTG mutagenesis conditions respectively.The DES mutagenic conditions is that DES concentration was 9 ?L/mL and the mutagenesis time was 20 min.The NTG mutagenic conditions was that NTG concentration was 0.6 mg/mL and the mutagenesis time was 15 min.After three rounds of mutagenesis and selection,five mutant strain were obtained with the increased keratinase activity and their keratinase activity were 13.4 U/m L,15.5 U/mL,14.2 U/mL,15.7 U/mL and 13 U/m L,which were increased by 26.4 %,46.2 %,33.96 %,48.1 %,22.6 % in comparison with that of its ancestor strain(10.6 U/mL).The optimization of protoplast preparation conditions by single factor experiments,results show that:the optimum conditions were 10 mg/mL lysozyme at 37�C for 30 min.The starting populations,obtained by DES and NTG mutagenesis,were subjected to recursive protoplast fusion,The positive colonies from fusing the protoplast fusion were screened.After three rounds of genome shuffling,six high keratinase activity strain was obtained.F3-5 and F3-6 were higher keratinase activity,its keratinase activity reached 68.3 U/mL and 68.7 U/mL and 7-fold in comparison with that of the parent strain.F3-5 and F3-6 mutant strain continuous 5 generation culture show they were genetic stability.